Crystal Violet Staining for Estimating Viral Infectious Titer in Cell Cultures

الملخص

Source: Frias-De-Diego and A., Crisci, E. Use of Crystal Violet to Improve Visual Cytopathic Effect-based Reading for Viral Titration using TCID50 Assays. J. Vis. Exp., (2022)

This video demonstrates a crystal violet staining method to visualize cytopathic effects in virus-infected cell cultures. The pattern of stained and unstained wells across serial dilutions enables rapid, cost-effective estimation of the viral infectious dose.

البروتوكول

1. Titration protocol

   NOTE: Use a cytopathic virus infecting adherent cells. For this demonstration, Influenza A Virus (IAV) of swine origin (A/California/07/2009/(H1N1)) and Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) Type 2, strain NC 1-7-4 were used.

   1. Titrate these viruses in 96 well plates for 7 days in a biosafety cabinet located in a Biosafety Level 2 (BSL-2) laboratory.
      1. To perform these titrations, seed 96 well plates with the required cell line. For PRRSV, use MA-104 cell line and for IAV use MDCK cell line. For the cell culture, use DMEM medium supplemented with 10% FBS, L-Glutamine and Penicillin-Streptomycin and grow the cells to confluency.
      2. Before infection, wash the cells using 200 µL of PBS.
      3. Dilute the virus stocks using 10 fold dilutions series by mixing 900 µL of media and 100 µL of virus. Make sure to properly vortex the tube to ensure proper mixing of the medium and virus and to avoid dilution errors.
      4. Mark the layout of the plate on the lid. Wash the wells with a 1x phosphate-buffered saline (PBS). Add 50 µL of the inoculum in the corresponding wells following previously described titration methods.
      5. Incubate at 37 °C in a 5% CO₂ incubator for 7 days.

2. Staining protocol

   NOTE: Cytopathic effect (CPE) was assessed via crystal violet staining.

   1. After the 7-day incubation, wash all the wells using 200 µL of 1x PBS twice.
   2. Fix the cells by adding 50 µL/well of 4% paraformaldehyde (PFA) in 1x PBS and incubate for 15 min at room temperature (RT).

   3. After incubation, wash the cells twice with 200 µL of 1x PBS. Then, add 50 µL/well of crystal violet diluted to 4% in water and incubate for 5 min at RT.

      NOTE: Crystal violet chemical stains the cells that remain attached to the plate at the time of fixation, leaving the sections of the well where the cells detached as unstained.

   4. Finally, aspirate crystal violet from the wells and optionally leave the plates to air dry for 2-5 min at RT or wash the plate with 200 µL of water to remove excess stain prior to visualization.
   5. Use mathematical methods to calculate the titer. Here, Karber formula and Muench formula were applied for PRRSV and IAV, respectively.

   6. For PRRSV, apply the Karber method:

      Titer (TCID50) = 10^{T + 1.3} where:

      

      In this formula d = negative log of the last dilution with complete positive virus response: five positive replicates; r = log of dilution range; N = number of replicates by dilution; n = number of wells with positive virus response on the next dilutions.

      In the case of IAV, use the Muench formula:

      

المواد

قائمة المواد المستخدمة في هذه المقالة

الاسم	الشركة	رقم فهرسي	التعليقات
96-well cell culture plates	Genesee	25-221	Clear, flat bottom
Crystal violet	Thermo Fisher	C581-25; C581-100
DMEM	Corning	10-017-CV
Fetal bovine serum	BioWest	S1480
Paraformaldehide	Thermo Fisher	J19943